Microbial diversity is important in a variety of contexts, in particular having implications for infectious diseases, bioremediation, and environmental engineering. Examination of the mechanisms underlying such diversity and its evolution are important for providing a roadmap that can lead to better understanding of the above-mentioned and other related areas. Several examples are available where mutualism in microbial communities can lead to the maintenance of microbial diversity. Fewer examples have been presented where some individuals provide protection to others in a population without a concomitant benefit being returned to the protector. Protection of sensitive members of a population against antimicrobials by resistant genotypes has been observed in biofilms. In these cases, spatial proximity to the protector/producer was a prerequisite, or at least an important component, for survival by otherwise sensitive individuals. We intend to extend these studies to shaking liquid or planktonic populations in order to examine the dynamics of such unrewarded protection or altruism. We have developed a family of mathematical models that have examined antibiotic resistance in such systems and have generated preliminary data which support some aspects of these models. Our results so far show that such altruists can provide frequency-dependent antibiotic resistance to other members in the population. Furthermore, frequency-dependent selection of the traits of these altruists promotes microbial diversity. In the current proposal, we will undertake more detailed experiments designed to test our model's predictions, and use the results obtained to further refine the models. Specifically, we will use competitions between near-isogenic Escherichia coli strains that are either sensitive to ampicillin or are resistant, by virtue of a plasmid-encoded ?-lactamase. Among those strains that are resistant, we will compare the outcome for those strains that only protect themselves from ampicillin versus those strains that can also provide protection to others in their vicinity, i.e., by destroying ampicillin nearby in the medium. These analyses will also be extended to competition experiments with greater relevance for natural clinical or human settings. For the latter analyses, the engineered E. coli strains described above will be competed against ampicillin-sensitive Enterococcus faecalis strains and, in a separate set of experiments, against a mixture of human fecal bacteria. The population dynamics of sensitive and resistant individuals in these trials will be explored. We predict that the altruistic E. coli will provide protection to their sensitive cohorts and thereby maintain diversity in a setting otherwise unfavorable for such diversity.